Tumor necrosis factor alpha (TNF-α) is a cytokine produced by numerous cell types, including monocytes and macrophages, that has been implicated in mediating shock and the pathophysiology of a variety of human diseases and disorders including sepsis, infections, autoimmune diseases, transplant rejection and graft-versus-host disease.
In an effort to counter the harmful effects mediated by human TNF-α, antibodies that bind to and neutralise human TNF-α have been sought as a means to inhibit TNF-α activity. Some of the earliest antibodies directed against human TNF-α were mouse monoclonal antibodies secreted from hybridoma cell lines prepared from lymphocytes harvested from mice immunized with human TNF-α. Although such antibodies were effective in binding to and neutralising human TNF-α, their use in in vivo therapy has been limited by problems associated with the administration of mouse antibodies to humans, in particular, elicitation of an unwanted immune response against the mouse antibody in a human, referred to as human anti-mouse antibody (HAMA) reactions.
In an attempt to overcome these problems, murine anti-human TNF-α antibodies have been genetically engineered to be more human-like. For example, human/mouse chimeric antibodies have been created in which antibody variable region sequences from the mouse genome are combined with antibody constant region sequences from the human genome. The chimeric antibodies exhibit the binding characteristics of the parental mouse antibody, and the effector functions associated with the human constant region. Although these chimeric antibodies have been used in human therapy, they still retain some murine sequences and therefore still may elicit anti-chimeric antibody reactions in human recipients, particularly when administered for prolonged periods thus limiting their therapeutic application.
Human monoclonal antibodies against human TNF-α have been developed using human hybridoma techniques. This approach, however, suffers from ethical, clinical and immunological limitations on immunization of human subjects.
It has been postulated that non-human primate antibodies will be tolerated in humans because they are structurally similar to human antibodies (Ehrlich P H et al., Human and primate monoclonal antibodies for in vivo therapy. Clin Chem. 34:9 pg 1681-1688 (1988)). Furthermore, because human antibodies are non-immunogenic in Rhesus monkeys (Ehrich P H et al., Rhesus monkey responses to multiple injections of human monoclonal antibodies. Hybridoma 1987; 6:151-60), it is likely that the converse is also applicable and primate antibodies will be non-immunogenic in humans.
Evolutionarily distant primates, such as New World primates, are not only sufficiently different from humans to allow antibodies against human antigens to be generated, but are sufficiently similar to humans to have antibodies similar to human antibodies so that the host does not generate an anti-antibody immune response when such primate-derived antibodies are introduced into a human. New World primates (infraorder-Platyrrhini) comprises at least 53 species commonly divided into two families, the Callithricidae and Cebidae. The Callithricidae consist of marmosets and tamarins. The Cebidae includes the squirrel monkey, titi monkey, spider monkey, woolly monkey, capuchin, night or owl monkey and the howler monkey.
Previous studies have characterised the expressed immunoglobulin heavy chain repertoire of the Callithrix jacchus marmoset (von Budingen H—C et al., Characterization of the expressed immunoglobulin IGHV repertoire in the New World marmoset Callithrix jacchus. Immunogenetics 2001; 53:557-563). Six IGHV subgroups were identified which showed a high degree of sequence similarity to their human IGHV counterparts. The framework regions were more conserved when compared to the complementarity determining regions (CDRs). The degree of similarity between C. jacchus and human IGHV sequences was less than between Old World primates and humans.
Domain Antibodies
Domain antibodies (dAb) are the smallest functioning binding units of antibodies and correspond to the variable regions of either the heavy (VH) or light (VL) chains of antibodies. Domain antibodies have a molecular weight of approximately 13 kDa, or less than one tenth the size of a full antibody.
Immunoglobulin light chains are referred to as either kappa or lambda light chains and the heavy chains as gamma, mu, delta, alpha or epsilon. The variable region gives the antibody its specificity. Within each variable region are regions of hypervariability, otherwise known as complementarity determining regions (CDRs) which are flanked by more conserved regions referred to as framework regions. Within each variable region are three CDRs and four framework regions.
In contrast to conventional antibodies, domain antibodies are well expressed in bacterial, yeast and mammalian systems. Their small size allows for higher molar quantities per gram of product, thus providing a significant increase in potency per dose. In addition, domain antibodies can be used as a building block to create therapeutic products such as multiple targeting dAbs in which a construct containing two or more variable domains bind to two or more therapeutic targets, or dAbs targeted for pulmonary or oral administration.